US3278394A - Method and composition for diagnosing glucose - Google Patents

Method and composition for diagnosing glucose Download PDF

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Publication number
US3278394A
US3278394A US296718A US29671863A US3278394A US 3278394 A US3278394 A US 3278394A US 296718 A US296718 A US 296718A US 29671863 A US29671863 A US 29671863A US 3278394 A US3278394 A US 3278394A
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glucose
color
composition
solution
test
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Expired - Lifetime
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US296718A
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English (en)
Inventor
Edward K Harvill
Ku Edmond Chiu-Choon
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Bayer Corp
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Miles Laboratories Inc
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Priority to US296718A priority Critical patent/US3278394A/en
Priority to CH894764A priority patent/CH439803A/de
Priority to SE8856/64A priority patent/SE304122B/xx
Priority to DEM61820A priority patent/DE1299445B/de
Priority to FR982677A priority patent/FR1401919A/fr
Priority to BE650845D priority patent/BE650845A/xx
Priority to NL6408380A priority patent/NL6408380A/xx
Application granted granted Critical
Publication of US3278394A publication Critical patent/US3278394A/en
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Expired - Lifetime legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose

Definitions

  • This invention relates to an improved diagnostic composition and particularly to a diagnostic composition useful in the quantitative determination of glucose in blood. More particularly, this invention is concerned with an improved liquid test system for accurately measuring the amount of glucose present in blood.
  • a number of the recently developed reagents and techniques involve the use of two enzymes and an indicator, which indicator is oxidized by hydrogen peroxide in the presence of peroxidase and undergoes a color reaction during such oxidation.
  • one particular reagent formulation for use in glucose determination comprises glucose oxidase, peroxidase, orthotolidine dihydrochloride, a buffer and gelatin as set forth, for example, in U.S. Patent No. 2,848,308 to Alfred H. Free, issued August 19, 1958, and assigned to the assignee of the present application.
  • Gelatin was incorporated into the ycomposition because it was found that its use resulted in the production of deep, sharply defined color, as distinguished from more poorly defined or inconclusive color which was produced by the same compositions in the absence of the gelatin.
  • an improved diagnostic composition for the quantitative determination of glucose in blood which offers the advantage of an extremely rapid color development and the maintenance of maximum color value for extended periods of time, for example up to about -two hours, as compared with a maximum of about seven minutes for the prior art compositions.
  • compositions of the present invention allow time for more accurate comparison of the developed color with a color chart, and it also allows time for ygradation of samples against each other directly. Such direct gradation is, of course, impossible, when previously known diagnostic compositions are used, because of the ⁇ shortness of the time during which the maximum color developed thereby can be maintained.
  • the composition of thisV invention comprises the enzyme glucose oxidase, anindicator oxidizable to produce an observable color change, an activator material capable. to catalyzing the oxidation of said indicator in the presence of hydrogen peroxide, and a protective colloid effective to stabilize the color produced by oxidation of said indicator.
  • Glucose oxidase catalyzes the conversion of 'glucose to gluconic acid and hydrogen peroxide in the presence of atmospheric oxygen.
  • the preferred activator material is the enzyme peroxidase.
  • a buffer is also preferably used to maintain the pH of the reactants at the site of reaction within the optimum range as hereinafter described.
  • composition of this invention may be prepared as a suspension or a solution and lyophilized, or it may be mixed dry and ground.
  • the ingredients are ground in the ratio desired in a mortar or other appropriate container allowing for good refining and mixing of the ingredients.
  • the powder is then weighed into a vial in the quantity desired and the vial is capped for storage.
  • lyophilization may readily be accomplished at temperatures of about from 0 C. to 20 C.
  • the color indicator there may be used any material which is oxidized in the presence of a peroxide and a catalytic activating substance, and which will undergo an observable color change such that the opitcal density of the indicator is proportional to the glucose concentration in the blood specimen being tested.
  • FIGURE 1 shows this relationship graphically using ortho-tolidine as the indicator and sodium alginate as the protective colloid.
  • Other indicators which may be used include, for example, bendidine, gum guaiac, 2,7-diaminouorene, o-dianisidine, leucoindophenols, -3,4-dihydroxyphenylalanine and guaiacol. Of these ortho-tolidine is preferred.
  • Various buffers may be used to provide the preferred pH range of from about pH 4.2 to about pH 4.8. At lower pHs enzymes are susceptible to denaturation,
  • Buffers which may be used include, for example, citrate, acetate, phosphate and tricarballylate as well as the tris buffers. These buffers are salts of organic acids with the amine tris (hydroxymethyl) aminomethane.
  • tris (hydroxymethyl) methylamine citrate sometimes called tris-citrate, tris-tricarballylate, tris-propionate, trisformate, tris-lactate, tris-malonate, tris-gluconate, trisglucuronate, tris-succinate, tris-tartrate and tris-aconitate.
  • tris-phosphate does not satisfy the requirement of being an amine salt of an organic acid tris (hydroxymethyl) methylamine phosphate may be used similarly.
  • the protective colloid of this invention may be any of a number of materials so characterized.
  • sodium alginate, gum arabic, gelatin, bovine serum albumin, carboxymethyl cellulose, agar or Carbowax have all been used satisfactorily to a greater or lesser extent.
  • sodium alginate and gum arabic have been found to give unexpectedly superior results and are therefore the preferred protective colloids of this invention.
  • the operative range of concentration of sodium alginate and gum arabic was found to be from 0.5 mg to 10.0 mg. per unit test of about 24.0 mg., and the optimum concentration of these protective colloids was found to be about 1.0 mg. per unit test of about ⁇ 24.0 mg.
  • EXAMPLE 2 By way of illustration of the rapid maximum color development and the maintenance of the maximum color value for an extended period of time, aqueous glucose solutions containing 40 and 80 mg. percent glucose were prepared and tested utilizing the composition prepared in accordance with Example l. The vial contents were dissolved in 6 ml. of water and 3 ml. of the glucose solutions added to the test solution. After mixing, the color of the solution was read by means of a spectrophotometer at 6200 Angstroms for a total period of 1 hour. The results of this test are shown in Table I.
  • FIGURE 2 shows a comparison between the test compositions of this invention utilizing sodium alginate as a protective colloid to stabilize color development and compositions which are otherwise the same but lack the protective colloid.
  • FIGURE 3 shows uniform color maintenance at all glucose concentrations from 30 mg. percent to 150y mg. percent as a result of the use of sodium alginate in the test composition.
  • a test composition for adding to and accurately detecting glucose in aqueous solution which consists of, in said aqueous solution, glucose, oxidase, perioxidase, a color indicator which is oxidized in the presence of a peroxide and perxoidase to produce an observable color change, a buffer for effecting a pH of from about 4.2 to about 4.8 at the reaction site when the test composition is contacted with material to be tested, and as a protective colloid effective to stabilize the color produced by oxidation of said indicator, sodium alginate.
  • test composition of claim 1 in the form of a powder.
  • test composition of claim 2 in the form of a lyophilized powder.
  • a method for accurately and quantitatively detecting glucose in solution which comprises adding an aqueous solution of a test composition consisting of glucose oxidase, peroxidase, a color indicator which is oxidized in the presence of a peroxide and peroxidase to produce an observable color change, a buffer for effecting a pH of from about 4.2 to about 4.8 in said solution and sodium alginate to said glucose solution and thereafter determining the amount of color formed in said solution which is indicative of the concentration of glucose present.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Emergency Medicine (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
US296718A 1963-07-22 1963-07-22 Method and composition for diagnosing glucose Expired - Lifetime US3278394A (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
US296718A US3278394A (en) 1963-07-22 1963-07-22 Method and composition for diagnosing glucose
CH894764A CH439803A (de) 1963-07-22 1964-07-08 Diagnostisches Nachweisreagens
SE8856/64A SE304122B (de) 1963-07-22 1964-07-21
DEM61820A DE1299445B (de) 1963-07-22 1964-07-21 Glukose-Diagnostikum
FR982677A FR1401919A (fr) 1963-07-22 1964-07-22 Composition d'analyse pour le diagnostic du glucose sanguin
BE650845D BE650845A (de) 1963-07-22 1964-07-22
NL6408380A NL6408380A (de) 1963-07-22 1964-07-22

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US296718A US3278394A (en) 1963-07-22 1963-07-22 Method and composition for diagnosing glucose

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US3278394A true US3278394A (en) 1966-10-11

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US296718A Expired - Lifetime US3278394A (en) 1963-07-22 1963-07-22 Method and composition for diagnosing glucose

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US (1) US3278394A (de)
BE (1) BE650845A (de)
CH (1) CH439803A (de)
DE (1) DE1299445B (de)
NL (1) NL6408380A (de)
SE (1) SE304122B (de)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3371019A (en) * 1965-01-08 1968-02-27 Searle & Co Diagnostic indicator
US4076502A (en) * 1975-02-14 1978-02-28 Miles Laboratories, Inc. Method, composition, and device for determining the specific gravity of a liquid
WO1987007718A1 (en) * 1986-06-02 1987-12-17 Lawrence Paul J Fecal occult blood test reagents and methods
US4818702A (en) * 1986-06-02 1989-04-04 Litmus Concepts, Inc. Fecal occult blood test reagent
US4820646A (en) * 1984-12-11 1989-04-11 Litmus Concepts, Inc. Fecal occult blood test method

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4098574A (en) * 1977-08-01 1978-07-04 Eastman Kodak Company Glucose detection system free from fluoride-ion interference

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2848308A (en) * 1955-12-05 1958-08-19 Miles Lab Composition of matter
US2912309A (en) * 1956-02-06 1959-11-10 Miles Lab Indicator for detecting glucose
US3042496A (en) * 1959-03-27 1962-07-03 Miles Lab Diagnostic composition
US3066081A (en) * 1961-05-05 1962-11-27 Edward S Rorem Means for detecting galactose
US3092465A (en) * 1960-03-25 1963-06-04 Miles Lab Diagnostic test device for blood sugar
US3123443A (en) * 1960-04-18 1964-03-03 Composition for diagnosing glucose

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1289712A (fr) * 1960-04-18 1962-04-06 Miles Lab Composition pour le diagnostic du glucose

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2848308A (en) * 1955-12-05 1958-08-19 Miles Lab Composition of matter
US2912309A (en) * 1956-02-06 1959-11-10 Miles Lab Indicator for detecting glucose
US3042496A (en) * 1959-03-27 1962-07-03 Miles Lab Diagnostic composition
US3092465A (en) * 1960-03-25 1963-06-04 Miles Lab Diagnostic test device for blood sugar
US3123443A (en) * 1960-04-18 1964-03-03 Composition for diagnosing glucose
US3066081A (en) * 1961-05-05 1962-11-27 Edward S Rorem Means for detecting galactose

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3371019A (en) * 1965-01-08 1968-02-27 Searle & Co Diagnostic indicator
US4076502A (en) * 1975-02-14 1978-02-28 Miles Laboratories, Inc. Method, composition, and device for determining the specific gravity of a liquid
US4820646A (en) * 1984-12-11 1989-04-11 Litmus Concepts, Inc. Fecal occult blood test method
WO1987007718A1 (en) * 1986-06-02 1987-12-17 Lawrence Paul J Fecal occult blood test reagents and methods
US4818702A (en) * 1986-06-02 1989-04-04 Litmus Concepts, Inc. Fecal occult blood test reagent

Also Published As

Publication number Publication date
DE1299445B (de) 1969-07-17
CH439803A (de) 1967-07-15
SE304122B (de) 1968-09-16
NL6408380A (de) 1964-10-26
BE650845A (de) 1964-11-16

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